Recombinant Human Lactate Dehydrogenase B/LDHB Protein, CF Summary
Ala2-Leu334, with N-terminal Met and 6-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl, TCEP and Glycerol.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 25 mM Tris, 100 mM NaCl, pH 7.5
- Recombinant Human LDHB (rhLDHB) (Catalog # 9205-HB)
- beta -Nicotinamide adenine dinucleotide, reduced disodium salt hydrate ( beta -NADH) (Sigma, Catalog # N8129), 20 mM stock in 0.1 M Sodium Borate, pH 9.0
- 100 mM Sodium Pyruvate (Gibco, Catalog # 11360)
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhLDHB to 0.4 ng/µL in Assay Buffer.
- Prepare a substrate mixture containing 1.6 mM beta -NADH and 4 mM sodium pyruvate in Assay Buffer.
- In a plate load 50 µL of 0.4 ng/µL rhLDHB, and start the reaction by adding 50 µL of substrate mixture. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of substrate mixture.
- Read plate at 340 nm (absorbance) in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol x (-1)|
|ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Using the extinction coefficient 6220 M-1cm-1
***Using the path correction 0.320 cm
Note: the output of many spectrophotometers is in mOD.
- rhLDHB: 0.020 µg
- beta -NADH: 0.8 mM
- Sodium pyruvate: 2 mM
Background: Lactate Dehydrogenase B/LDHB
A hallmark of most cancer cells is an altered metabolism involving a shift to aerobic glycolysis with lactate production coupled with a higher uptake of glucose as the main source of energy. Lactate dehydrogenase (LDH) is key to this shift by catalyzing the formation of lactate by reducing pyruvate with NADH, which also generates NAD(+) that is essential for the continuity of glycolysis (1). Inhibiting LDH activity has an anti-proliferative effect on cancer cells (2). It may reverse the resistance of tumor cells to conventional chemo- and radiotherapy. Recent research has renewed interest in LDH as an anticancer drug target (3). LDH enzymes have three homologous subunits LDHA, LDHB and LDHC. The combination of these three results to multiple isozymes, including LDH-M and LDH-H. Mutations in LDHB cause lactate dehydrogenase B deficiency, a condition with no deleterious effects on health (4).
- Faloppi L. et al. (2016) Sci Rep. 6:24136.
- Ghosh, M. et al. (2016) Chem. Commun. 52:2401.
- Augoff, K. et al. (2015) Cancer Lett. 358:1.
- Maekawa M. et al. (1993). Hum. Genet. 91:423.
- Maekawa M. et al. (1990). Biochem. Biophys. Res. Commun. 168:677.
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